DESCRIPTION: (Investigator's abstract) The platelet membrane glycoprotein Ib-IX complex (GP Ib-IX) plays an important role in the initial platelet adhesion to injured vascular wall. By binding to subendothelial von Willebrand factor (vWF), GP Ib-IX not only mediates the physical adherence of platelets, but also initiates an activation signal. This activates the platelet integrin alphaIIbbeta3 which subsequently mediates platelet spreading and aggregation. Overall objective of the proposed studies is to determine the mechanism of GP Ib-IX-mediated signaling. We have found that the cytoplasmic domain of GP Ib-IX interacts with an important intracellular signaling molecule, zeta-form 14-3-3 protein (14-3-3zeta). 14-3-3zeta functions as a phosphorylation-dependent adapter that links kinases and other molecules, forming signaling complex. 14-3-3zeta regulates functions of c-Raf and several isoforms of protein kinase C. The proposed studies will test the hypothesis that 14-3-3zeta is a key link between GP Ib-IX and the intracellular signaling pathway. In supporting studies, we have characterized the structural basis of GP Ib-IX-14-3-3 interaction, and developed a series of 14-3-3zeta deletion mutants containing GP Ibalpha binding site or raf kinase binding site. Further, we have shown that we are able to reconstitute GP Ib-IX-mediated integrin activation in a recombinant DNA stable expression model in mammalian cells. This will enable us to apply molecular biological techniques (which cannot be used in the anucleate platelet) to specifically examine the roles of 14-3-3zeta and other signaling molecules in GP Ib-IX-mediated signaling. We propose to (1) reconstitute and characterize the GPI b-IX-mediated signaling pathway in a mammalian cell expression model; (2) determine the roles of 14-3-3zeta and GP Ib-IX-mediated signaling using the mammalian cell expression model and mutants of 14-3-3 and GP Ib; (3) identify downstream pathways of 14-3-3 mediated GPI b-IX signaling; and (4) determine the roles of phosphorylation of GP Ib-IX in the regulation of the interaction between GP Ib-IX and 14-3-3zeta in the GP Ib-IX-mediated signaling.